This invention relates to novel polynucleotides, polypeptides encoded by them, use of the polynucleotides and polypeptides, and a method for producing them. More particularly, the present invention relates to Calcineurin (CN)-binding polypeptides.
The calcium/calmodulin-dependent serine/threonine phosphatase, Calcineurin (CN) (Masuda, E. S. et al., 1999, Cell. Signaling 10:599), is a heterodimeric protein composed of a calmodulin-binding catalytic subunit, CNA, and a Ca2+-binding regulatory subunit, CNB (Kincaid, R. 1993, Adv. Second Messenger Phosphoprotein Res. 27:1). CN is a target of the immunosuppressive drugs cyclosporin A (CSA) and FK506, which block T cell function by preventing transcriptional activation of cytokine genes. It has been suggested that CN plays an essential role in calcium-dependent dephosphorylation signal transduction pathways and subsequently leads to production of cytokines in T cells (Crabtree, G. R., and N. A. Clipstone, 1994, Annu. Rev. Biochem. 63:1045). Further studies have revealed that the potential of CN to regulate the expression of cytokine genes is largely due to effects on activation of a transcription factor termed nuclear factor of activated T cells (NF-AT) (Rao, A. et al., 1997, Annu. Rev. Immunol. 15:707; Masuda, E. S. et al., 1999, Cell. Signaling 10:599).
Currently, five NF-AT family members (NF-AT1/NF-ATp, NF-ATc/NF-AT2, NF-ATx/NF-AT4/NF-ATc3, NF-AT3, and TonE-BP/NF-AT5) have been identified, and they share functional and structural similarities (Hoey, T. et al., 1995, Immunity 2:461; Masuda, E. S. et al., 1995, Mol. Cell. Biol. 15:2697; McCaffrey, P. G. et al., 1993, Science 262:750; Northrop, J. P. et al., 1994, Nature 369:497; Miyakawa, H. et al., 1999, Proc. Natl. Acad. Sci. USA 96: 2538-42; Lopez-Rodriguez, C. et al., 1999, Proc. Natl. Acad. Sci. USA 96: 7214-9). The NF-AT complex is composed of at least two components. Both activation of the protein kinase C/Ras pathway and the elevated level of intracellular calcium are required for activation of this complex. The former is responsible for formation of the AP1 (activating protein-1) complex, as the nuclear components of NF-AT, while the latter leads to translocation of NF-AT from the cytoplasm to the nucleus, where it binds with AP1 at IL-2 promoter NF-AT sites (Crabtree, G. R., and N. A. Clipstone, 1994, Annu. Rev. Biochem. 63:1045; Rao, A. 1994, Immunol. Today 15:274). Thus, nuclear transport is a critical step that allows NF-AT to function in the nucleus. CN has been shown to dephosphorylate NF-AT, the result being nuclear translocation of NF-AT (Karen, T.-Y. S. et al., 1995,
Natl. Acad. Sci. USA 92:11205), which can be inhibited by and FK506 (Bierer, B. E. 1994, Chem. Immunol. 59:128).
NF-AT protein is functionally divided into three domains. First, the Rel similarity domain (RSD) has a high sequence homology among different family members. It is responsible for DNA binding and cooperatively interacts with AP1 proteins (Hoey, T. et al., 1995, Immunity 2:461; McCaffrey, P. G. et al., 1993, Science 262:750; Jain, J. et al., 1995, J. Biol. Chem. 270:4138). In addition, one of the two putative conserved nuclear localization signals (NLSs) present in NF-AT family members is located within RSD (Hoey, T. et al., 1995, Immunity 2:461; Masuda, E. S. et al., 1995, Mol. Cell. Biol. 15:2697; McCaffrey, P. G. et al., 1993, Science 262:750; Northrop, J. P. et al., 1994, Nature 369:497). Second, a C-terminal domain eliciting less sequence homology has been reported to bear a transactivation motif (Imamura, R. et al., 1998, J. Immunol. 161:3455). The third domain showing homology among NF-AT proteins is the N-terminal domain. Several conserved motifs, such as SP boxes that are rich in serines and prolines (Masuda, E. S. et al., 1995, Mol. Cell. Biol. 15:2697), CN-regulated inhibitory (CRI) sequence/serine-rich region (SRR) (Masuda, E. S. et al., 1997, Mol. Cell. Biol. 17:2066; Beals, C. R. et al., 1997, Genes Dev. 11:824), and another functional NLS (Beals, C. R. et al., 1997, Genes Dev. 11:824; Luo, C. et al., 1996, Proc. Natl. Acad. Sci. USA 93:8907), have been identified within the N-terminal domain. The conserved serine residues in the SRR motif were found to be constitutively phosphorylated by cellular kinases and can be dephosphorylated by CN (Beals, C. R. et al., 1997, Genes Dev. 11:824). Deletion of CRI in human NF-ATx1 (hNF-ATx1) or mutation of serines in the SRR motif of NF-ATc led to the constitutive nuclear translocation of either hNF-ATx1or NF-ATc (Masuda, E. S. et al., 1997, Mol. Cell. Biol. 17:2066; Beals, C. R. et al., 1997, Genes Dev. 11:824). Furthermore, at least two conserved NLSs have been reported to be essential for the nuclear translocation of NF-ATc; one NLS located in RSD is associated with the majority of phosphorylated serines in SRR (Beals, C. R. et al., 1997, Genes Dev. 11:824). Thus, NLS is probably masked by these phosphorylated serine residues. Both the domain interacting with CN and residues dephosphorylated by CN have been mapped within the N-terminus of NF-AT (Masuda, E. S. et al., 1995, Mol. Cell. Biol. 15:2697; Beals, C. R. et al., 1997, Genes Dev. 11:824; Luo, C. et al., 1996, Proc. Natl. Acad. Sci. USA 93:8907), suggesting that the N-terminal domain of NF-AT is a target of CN action involved in major activities of the Ca2+ signaling pathway and is important for the nuclear localization of NF-AT. However, detailed interaction between CN and NF-AT has not been clarified.
Structural and functional analyses of the N-terminal domain of murine NF-ATx1 (mNF-ATx1), (Liu, J. et al., 1997, Mol. Biol. Cell. 8:157), a member of the NF-AT family, have defined two distinct CN binding regions (CNBRs), CNBR1 and CNBR2, which are located in the region preceding the SP boxes of serine/proline-rich sequences and the region between the SP boxes and Rel similarity domain, respectively. Each of the two CN binding regions has the capacity to independently bind CN. The binding of mNF-ATx1 to CN was abolished by deletion of these two regions, yet was unaffected by the individual deletion. In contrast, the nuclear translocation of mNF-ATx1 was much reduced when only CNBR2 was removed. Luciferase assay revealed that both regions are required for mNF-ATx1-dependent activation of the murine IL-2 promoter. Most importantly, recombinant CNBR2 bound CN with a higher affinity, and when expressed in Jurkat cells, it functioned as a dominant negative mutant that prevented the transcription driven by exogenous mNF-ATx1, probably by interfering with the function of CN. The present invention revealed important features of the interaction of mNF-ATx1 with CN via the CN binding region, and light was shed on a structure-function model of mNF-ATx1 proteins. The finding that one of two CN binding regions acts as an inhibitor of mNF-ATx1 opens the way for development of immunosuppressive agents. The present invention provides a new opportunity for pharmacological intervention with Ca2+-dependent signaling events.
In one aspect, the invention relates to CN binding polypeptides and DNAs encoding them, and methods for their production.
Another aspect of the invention relates to methods for using polypeptides and polynucleotides of the invention. In particular, the present invention relates to a method for screening a compound that inhibits interation between NF-AT and CN using the polypeptides of the present invention.
Still another aspect of the present invention is pharmaceutical compositions comprising a polypeptide of the present invention or a compound isolable by the above screening method. The pharmaceutical compositions can be used to inhibit interaction between NF-AT and CN. CN/NF-AT signal transduction is involved in induction of an immune response. Immunoreaction can thus be suppressed by inhibiting interaction between NF-AT and CN. The pharmaceutical compositions of this invention are especially useful for suppressing rejection after transplantation of organs and treating or preventing autoimmune diseases. The polypeptides of this invention are useful for preventing hypercardia and hypertrophy of the vascular wall.
More specifically, the present invention relates to:
(1) a polypeptide having Calcineurin-binding activity selected from the group consisting of:
(a) polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 1 (CNBR1: positions 25 to 142 of mNF-ATx1) or SEQ ID NO: 2 (CNBR2: positions 321 to 406 of mNF-ATx1);
(b) polypeptides corresponding to the polypeptides of (a) contained in NF-Atx family proteins;
(c) polypeptides of (a) or (b) in which one or more amino acids are added, deleted, substituted, and/or inserted; and
(d) fusion polypeptides comprising a polypeptide of (a), (b) or
(c) and one or more other polypeptides;
(2) a DNA encoding the polypeptide of (1);
(3) a vector comprising the DNA of (2);
(4) a transformant carrying the DNA of (2) or the vector of (3);
(5) a method for producing the polypeptide of (1), the method comprising culturing the transformant of (4), and recovering the expressed polypeptide from the transformant or the culture supernatant;
(6) a method for screening a compound that inhibits the interaction between Calcineurin and NF-AT, the method comprising:
(a) contacting the polypeptide of (1) with Calcineurin in the presence or absence of a sample;
(b) detecting the binding activity of the polypeptide to Calcineurin; and
(c) selecting a compound that reduces the binding activity compared to the binding activity detected in the absence of a sample;
(7) a compound isolable by the screening method of (6);
(8) a pharmaceutical composition comprising the compound of (7) as an active ingredient;
(9) a pharmaceutical composition comprising the polypeptide of (1) as an active ingredient;
(10) a method of suppressing immune, the method comprising administering the pharmaceutical composition of (8) to a patient in need of immunosuppression; and
(11) a method of suppressing immune, the method comprising administering the pharmaceutical composition of (9) to a patient in need of immunosuppression.
(12) a method of preventing the hypertrophy of cardiac smooth muscle or vascular smooth muscle, the method comprising administering the pharmaceutical composition of (8) to a patient.
(13) a method of preventing the hypertrophy of cardiac smooth muscle or vascular smooth muscle, the method comprising administering the pharmaceutical composition of (9) to a patient.